Method for designing probe in dna microarray, and dna microarray provided with probe designed thereby

ABSTRACT

Provided is a probe to be used in a DNA microarray having an excellent detection rate of a polymorphism such as SNP contained in genomic DNA. A method for designing a probe according to the invention includes the steps of: specifying one or more regions covering at least a part of fragments flanked by restriction enzyme recognition sites recognized by a restriction enzyme, contained in genomic DNA derived from an organism to be tested; and designing a probe for the specified one or more regions for detecting the fragment in the organism to be tested.

TECHNICAL FIELD

The present invention relates to a method for designing a probe used ina DNA microarray for detecting, for example, a mutation in genomic DNA,a DNA microarray having a probe designed by the method, and a method fordetecting a mutation using the DNA microarray.

BACKGROUND ART

Of the polymorphisms represented by a single nucleotide polymorphism(SNP), there is a polymorphism that can be used as a mutationcharacterizing a variation in a homogenous organism. More specifically,a predetermined variation in a homogenous organism can be distinguishedfrom other variations by detecting and identifying a specific mutationsuch as a polymorphism in genomic DNA. Furthermore, a variation of anorganism to be tested can be specified by detecting and identifying themutation.

As a method for detecting such a mutation in genomic DNA, a method ofdirectly determining a sequence of a mutation site, a method of using arestriction enzyme fragment length polymorphism (RFLP), a method ofusing an amplification fragment length polymorphism (AFLP) and the likeare known. In addition, a method of analyzing a variation based onidentification of a polymorphism using a DNA microarray called as a DArT(Diversity Array Technology) method (Nucleic Acids Research, 2001, Vol.29, No. 4, e25) is known.

A method for preparing a DNA microarray for use in the DArT method isshown in FIG. 9. First, genomic DNA is extracted from a predeterminedorganism species and fractionated with restriction enzyme A andrestriction enzyme B. Next, to the both ends of each of the genomic DNAfragments obtained by the restriction enzyme treatment, an adaptor isconnected and each of the genomic DNA fragments is cloned into a vector.Next, using a primer capable of hybridizing with the adaptor, genomicDNA fragments are amplified by PCR. Then, genomic DNA fragmentsamplified are spotted on a substrate as a probe to prepare a DNAmicroarray.

Using the DNA microarray thus prepared, a variation of a organismspecies to be tested can be analyzed. First, genomic DNA is extractedfrom an organism to be tested, and fractionated with restriction enzymeA and restriction enzyme B that are used for preparing the DNAmicroarray. To the genomic DNA fragments, an adaptor is connectedsimilarly in the preparation of the DNA microarray and the resultantfragments are amplified by PCR. The amplified genomic DNA fragments aretagged with a fluorescent label etc. and hybridized with the probespotted on the DNA microarray. Based on the presence or absence ofhybridization of the labeled genomic DNA fragment with the probedetected, a difference between the predetermined organism species usedin preparation of the DNA microarray and the organism species to betested can be analyzed.

SUMMARY OF INVENTION Technical Problem

According to the DArT method, the diversity of an organism species canbe determined in a genotype level in the genomic DNA by using the DNAmicroarray prepared as mentioned above. However, the DNA microarrayprepared as mentioned above has a problem in that the detection abilityof a probe, which is defined as a region flanked by restriction enzymerecognition sites, is not sufficient. More specifically, even if agenomic DNA fragment derived from an organism species to be testedcontains a small mutation such as SNP, the genomic DNA fragment mayoften hybridize with the probe of the DNA microarray. In other words,the DArT method has a detection limit, that is, detection cannot be madeunless a mutation such as a polymorphism is present in a restrictionenzyme recognition site or deletion of several hundreds of base pairs ispresent.

Then, in the aforementioned circumstances, the present invention isdirected to providing a method for designing a probe of a DNA microarrayhaving an excellent detection rate of a polymorphism such as SNPcontained in genomic DNA, a DNA microarray having a probe designed bythe method and a method for detecting a mutation using the DNAmicroarray.

Solution to Problem

In the aforementioned circumstances, the present inventors have madeintensive studies and conceived a method for designing a probe capableof detecting even a small mutation such as SNP in the genomic DNA withan excellent sensitivity, and a method of detecting a mutation by usinga DNA microarray having the probe immobilized thereto.

The present invention includes the followings.

More specifically, the method for designing a probe according to thepresent invention including the steps of: specifying one or more regionshaving a shorter nucleotide length than fragments flanked by restrictionenzyme recognition sites recognized by a restriction enzyme, containedin genomic DNA derived from a target organism, and covering at least oneportion of the genomic DNA fragments; and designing the specified one ormore regions as a probe for detecting the fragment in an organism to betested.

The one or more regions can be specified by performing the followingsteps:

(1 a) extracting the genomic DNA;

(1 b) digesting the extracted genomic DNA with the restriction enzyme;

(1 c) connecting an adaptor to the genomic DNA fragments obtained thestep (1 b);

(1 d) amplifying the genomic DNA fragments using a primer capable ofhybridizing to the adaptor;

(1 e) sequencing the amplified genomic DNA fragment; and

(1 f) determining the one or more regions based on the nucleotidesequence.

In the step (1 b) herein, the genomic DNA may be digested with one ormore restriction enzymes. Furthermore, in the step (1 c), the adaptorused preferably has a complementary sequence to a protruding end of thegenomic DNA fragments obtained the step (1 b). Moreover, the region tobe determined in the step (1 f) has, for example, a 20 to 10000nucleotide length, preferably, a 100 to 8000 nucleotide length and morepreferably, a 200 to 6000 nucleotide length.

Furthermore, the one or more regions can be specified using nucleotidesequence data on the genomic DNA by performing the following steps:

(2 a) searching the nucleotide sequence data on the genomic DNA for therestriction enzyme recognition sequence to specify the nucleotidesequence of the genomic DNA fragments obtained by digesting the genomicDNA with the restriction enzyme; and

(2 b) determining the one or more regions based on the specifiednucleotide sequence.

Herein, the region determined in the step (2 b) has, for example, a 20to 10000 nucleotide length, preferably, a 100 to 8000 nucleotide length,and more preferably, a 200 to 6000 nucleotide length.

Furthermore, the one or more regions can be determined by performing thefollowing steps:

(3 a) extracting the genomic DNA;

(3 b) digesting the extracted genomic DNA with the restriction enzyme;

(3 c) connecting an adaptor to the genomic DNA fragments obtained in thestep (3 b);

(3 d) amplifying the genomic DNA fragments using a primer capable ofhybridizing to the adaptor;

(3 e) digesting the amplified genomic DNA fragment with anotherrestriction enzyme; and

(3 f) separating the DNA fragments obtained by digestion in the step (3e) as probes.

Furthermore, in the method for designing a probe according to thepresent invention, a fragment flanked by the restriction enzymerecognition sites may be a fragment flanked by more than one restrictionenzyme having different recognition sequences.

In the step (3 b) herein, the genomic DNA may be digested with one ormore restriction enzymes. Furthermore, in the step (3 c), the adaptorused preferably has a complementary sequence to a protruding end of thegenomic DNA fragment obtained the step (1 b).

On the other hand, the DNA microarray according to the present inventionis prepared by immobilizing a probe designed by the aforementionedmethod for designing a probe according to the present invention on acarrier. Particularly, in the DNA microarray according to the presentinvention, the probe is preferably synthesized on a carrier based on thesequence data.

On the other hand, a method for detecting a mutation using the DNAmicroarray according to the present invention is a method of detecting amutation in a genomic DNA derived from a target organism to be tested byusing the aforementioned DNA microarray according to the presentinvention. Particularly, a mutation detection method using the DNAmicroarray according to the present invention includes the followingsteps:

extracting a genomic DNA derived from a target organism to be tested;

digesting the genomic DNA with a restriction enzyme having the samerecognition sequence as the restriction enzyme used in the method fordesigning a probe according to the present invention;

connecting an adaptor to the genomic DNA fragments obtained by therestriction enzyme treatment;

amplifying the genomic DNA fragments using a primer capable ofhybridizing to the adaptor; and

detecting a hybrid of the genomic DNA fragment with the probe bybringing the amplified genomic DNA fragment into contact with the DNAmicroarray according to the present invention.

Herein, in the step of digesting the genomic DNA with the restrictionenzyme, the genomic DNA may be digested with one or more restrictionenzymes similarly to the method for designing a probe. Furthermore, inthe step of connecting the adaptor, as the adaptor, one having acomplementary sequence to a protruding end of the genomic DNA fragmentobtained in the step of digesting the genomic DNA with the restrictionenzyme is preferably used. Moreover, the step of amplifying the genomicDNA fragment may further have a step of adding a labeling molecule to anamplified genomic DNA fragment or may have a step of allowing thegenomic DNA fragment to incorporate a labeling molecule when the genomicDNA fragment is amplified.

The specification of the present invention incorporates the contentdescribed in the specification and/or drawings of JP Application No.2009-283430 A, based on which the priority of the present application isclaimed.

Advantageous Effects of Invention

According to the present invention, it is possible to provide a methodfor designing a probe having an excellent detection rate of apolymorphism such as SNP contained in genomic DNA, for use in a DNAmicroarray. Furthermore, according to the present invention, it ispossible to provide a DNA microarray having an excellent detection rateof a polymorphism such as an SNP contained in a genomic DNA and a methodfor detecting a mutation by use of the DNA microarray.

Application of the present invention enables to analyze, i.e., determineand identify, an organism species based on a genotype, although it hasbeen difficult to detect it by a conventional method.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a flow chart schematically showing a method for designing aprobe to which the present invention is applied.

FIG. 2 is a flow chart schematically showing another method fordesigning a probe to which the present invention is applied.

FIG. 3 is a flow chart schematically showing further another method fordesigning a probe to which the present invention is applied.

FIG. 4 is a flow chart schematically showing a step of detecting amutation by using a DNA microarray having a probe designed by applyingthe present invention.

FIG. 5 is a characteristic view showing alignment of A_(—)1 and A_(—)2and the site of the designed probe.

FIG. 6 is a characteristic view showing alignment of B_(—)1 and B_(—)2and the site of the designed probe.

FIG. 7 is a characteristic graph showing the relationship between therate of mutation introduced in a probe and the intensity of a signaldetected.

FIG. 8 is a characteristic graph showing the relationship between theratio of mutation site probe prepared in Example 3 and the ratio of thesequence data in which a mutation was detected.

FIG. 9 is a characteristic view schematically showing a step ofpreparing a DNA microarray used in a conventional DArT method.

DESCRIPTION OF EMBODIMENTS

Now, the method for designing a probe for use in the DNA microarrayaccording to the present invention, a DNA microarray having a probedesigned by the method for designing a probe, and a method of detectinga mutation by use of the DNA microarray, will be more specificallydescribed, referring to the drawings.

Method for Designing a Probe

The probe to be designed in the present invention is preferably appliedto, particularly, a so-called oligonucleotide microarray. Theoligonucleotide microarray is a microarray, which is prepared bysynthesizing an oligonucleotide having a desired nucleotide sequence ona carrier and using the oligonucleotide as a probe. The synthesizedoligonucleotide used as a probe has, for example, a 20 to 100 nucleotidelength, preferably a 30 to 90 nucleotide length, and more preferably a50 to 75 nucleotide length.

Note that, the probe designed in the present invention may be applied toa microarray, which is prepared by spotting a synthesizedoligonucleotide having the aforementioned nucleotide length onto acarrier, similarly to so-called Stanford-type microarray.

More specifically, the probe designed in the present invention can beapplied to any microarray as long as it is conventionally known.Therefore, the probe designed in the present invention can be applied toa microarray using a flat substrate formed of glass and silicone etc.,as a carrier and to a beads array using micro-beads as a carrier.

More specifically, in the method for designing a probe according to thepresent invention, first, genomic DNA is extracted from a predeterminedorganism, as shown in FIG. 1 (Step 1 a). As the organism, any one of amicroorganism such as a bacterium and a fungus, an insect, a plant andan animal may be used. Note that, the method for designing a probe shownin FIG. 1 is preferably applied to the case of using an organism whosegenomic DNA nucleotide sequence data has not yet been elucidated.Furthermore, a method of extracting genomic DNA is not particularlylimited and a method known in the art can be used.

Next, the extracted genomic DNA is digested with one or more restrictionenzymes (Step 1 b). In the example shown in FIG. 1, genomic DNA isdigested with two types of restriction enzymes, restriction enzyme A andrestriction enzyme B, which are sequentially used in this order. Therestriction enzymes used herein are not particularly limited. Forexample, PstI, EcoRI, HindIII, BstNI, HpaII and HaeIII can be used.Particularly, the restriction enzyme can be appropriately selected inconsideration of an appearance frequency of a recognition sequence suchthat genomic DNA is completely digested into genomic DNA fragmentshaving a 20 to 10000 nucleotide length. Furthermore, in the case wheremore than one restriction enzyme is used, it is preferred that after allrestriction enzymes are applied, the genomic DNA fragments of a 200 to6000 nucleotide length remain. Moreover, when more than one restrictionenzyme is used, the order of supplying restriction enzymes to treatmentis not particularly limited. Furthermore, when common treatmentconditions (solution composition, temperature etc.,) are used, more thanone restriction enzyme may be used in the same reaction system. Todescribe more specifically, in the example shown in FIG. 1, genomic DNAis digested by using restriction enzyme A and restriction enzyme B inthis order; however, restriction enzyme A and restriction enzyme B maybe simultaneously used in a same reaction system to digest genomic DNA.Alternatively, restriction enzyme B and restriction enzyme A may be usedin this order to digest genomic DNA. Furthermore, the number ofrestriction enzymes to be used may be 3 or more.

Next, to genomic DNA fragments treated by the restriction enzyme, anadaptor is connected (Step 1 c). The adaptor herein is not particularlylimited as long as it can connect to both ends of each of the genomicDNA fragments obtained by the aforementioned restriction enzymetreatment. As the adaptor, for example, one having a single strandcomplementary to a protruding end (sticky end) formed at both ends ofgenomic DNA fragments by restriction enzyme treatment, and having aprimer binding sequence to which a primer to be used in theamplification treatment (specifically described later) can hybridize,can be used. Furthermore, as the adaptor, one having a single strandcomplementary to the protruding end (sticky end) and having arestriction enzyme recognition site for use in cloning into a vector.

Furthermore, in the digestion of the genomic DNA with more than onerestriction enzyme, more than one adaptor can be prepared for usecorresponding to the restriction enzymes. More specifically, in thedigestion of the enomic DNA with more than one restriction enzyme, morethan one protruding end generates. To correspond to the more than oneprotruding end, more than one adaptor having a single strandcomplementary thereto can be used. At this time, the more than oneadaptor corresponding to the more than one restriction enzyme may have acommon primer binding sequence such that a common primer can hybridizeor may have different primer binding sequences such that differentprimers can hybridize.

Furthermore, in the digestion of genomic DNA with more than onerestriction enzyme, an adaptor can be prepared for use corresponding toone restriction enzyme selected from the more than one restrictionenzyme used or corresponding to a part of the restriction enzymes used.

Next, a genomic DNA fragment having an adaptor added to the both ends isamplified (Step 1 d). When the adaptor having a primer binding sequenceis used, the genomic DNA fragment can be amplified by use of a primercapable of hybridizing with the primer binding sequence. Alternatively,the genomic DNA fragment having an adaptor added thereto is cloned intoa vector by use of the adaptor sequence. In this case, the genomic DNAfragment can be amplified by use of a primer capable of hybridizing to apredetermined region of the vector. Note that, as an amplificationreaction of a genomic DNA fragment by use of a primer, for example, PCRcan be used.

Furthermore, in the case where genomic DNA is digested with more thanone restriction enzyme and more than one adaptor corresponding to therestriction enzymes are connected to the genomic DNA fragments, theadaptors will be connected to all genomic DNA fragments obtained bytreatment using more than one restriction enzyme. In this case, allgenomic DNA fragments obtained can be amplified by a nucleic acidamplification reaction using a primer binding sequence contained in eachof the adaptors.

Alternatively, in the digestion of the genomic DNA with more than onerestriction enzyme and the connection to the genomic DNA fragment anadaptor corresponding to one restriction enzyme selected from the morethan one restriction enzyme used or corresponding to a part of therestriction enzymes used, only a genomic DNA fragment, of the obtainedgenomic DNA fragments, having a recognition sequence of the selectedrestriction enzyme at both ends can be amplified.

Next, the amplified genomic DNA fragment is sequenced (Step 1 e), one ormore regions having a shorter nucleotide length than the genomic DNAfragments and covering at least a part of the genomic DNA fragments arespecified; and a probe for the one or more regions specified aredesigned for detecting the amplified genomic DNA fragment of an organismto be tested (Step 1 f). A method for sequencing a genomic DNA fragmentis not particularly limited. A method known in the art employing theSanger method etc. and a DNA sequencer can be used.

In the steps (Steps 1 e and 1 f), one or more regions having a shorternucleotide length than the amplified genomic DNA fragments are designedas a probe(s) for detecting the genomic DNA fragment. Herein, in thecase where more than one region of a predetermined genomic DNA fragmentare used for designing, detection of the genomic DNA fragment using morethan one probe is intended. Furthermore, a single region may be selectedfrom a genomic DNA fragment and designed, whereas a predetermined number(two or more) of regions may be selected from another genomic DNAfragment and designed. In short, the number of regions to be designedmay differ from one genomic DNA fragment to another. As the regions tobe designed herein, those having, for example, a 20 to 10000 nucleotidelength, preferably, a 100 to 8000 nucleotide length, and morepreferably, a 200 to 6000 nucleotide length are used, as mentionedabove. Furthermore, in the case where more than one region is designed,the adjacent regions may be overlapped with each other or may have aninterval of several nucleotides between them.

Particularly, more than one region is preferably set so as to cover theentire region of the sequenced genomic DNA fragment. In this case, morethan one probe responds to a genomic DNA fragment obtained by arestriction enzyme treatment from genomic DNA derived from apredetermined organism to detect the genomic DNA fragment by these morethan one probe.

In the meantime, the method for designing a probe according to thepresent invention is not limited to a method including a step ofdigesting genomic DNA with a restriction enzyme(s) as mentioned above,and genomic data of a target organism, as shown in FIG. 2 may be used.

In the method shown in FIG. 2, first, nucleotide sequence data on thegenome derived from a target organism is obtained (Step 2 a). Thenucleotide sequence data on the genome can be obtained from varioustypes of data bases known in the art. The data base is not particularlylimited; however, DDBJ data base provided by the DNA Data Bank of Japan,EMBL data base provided by the European Bioinformatics Institute,Genbank data base provided by the National Center for BiotechnologyInformation, the KEGG data base provided by the Kyoto Encyclopedia ofGenes and Genomes or data base integrated of these data bases can beappropriately used.

In this method, next, the nucleotide sequence data on the genomic DNAobtained is searched for the recognition sequence of the restrictionenzyme(s) as mentioned above (Step 2 a). The nucleotide sequences ofgenomic DNA fragments which will be obtained by digesting theaforementioned genomic DNA with the aforementioned restriction enzyme(s)are specified. The recognition sequence to be searched for herein is arestriction enzyme(s) corresponding to the restriction enzyme(s) used inthe method shown in FIG. 1. More specifically, in this step, recognitionsequences of one or more restriction enzymes are searched for.

Next, based on the nucleotide sequence of the determined genomic DNAfragment, one or more regions covering at least a part of the genomicDNA fragment are determined (Step 2 b). In the step (Step 2 b), one ormore regions having a shorter nucleotide length than the sequencedgenomic DNA fragments are designed as a probe for detecting the genomicDNA fragment. Herein, if more than one region is designed for apredetermined genomic DNA fragment, detection of the genomic DNAfragment using more than one probe is intended. Furthermore, a singleregion may be selected from a genomic DNA fragment and designed, whereasa predetermined number (2 or more) of regions may be selected fromanother genomic DNA fragment and designed. In short, the number ofregions to be designed may differ from one genomic DNA fragment toanother. As the region to be designed herein, those having, for example,a 20 to 100 nucleotide length, preferably a 30 to 90 nucleotide length,and more preferably a 50 to 75 nucleotide length are used, as mentionedabove.

Furthermore, more than one region is preferably set so as to cover theentire region of the sequenced genomic DNA fragment. In this case, morethan one probe responds to genomic DNA fragments obtained by arestriction enzyme treatment from genomic DNA derived from apredetermined organism to detect the genomic DNA fragment by these morethan one probe.

In the meantime, the method for designing a probe according to thepresent invention may be a method having neither a step of sequencingnor a step of obtaining nucleotide sequence data using a database andincluding a step of digesting the genomic DNA fragment with a furtherdifferent restriction enzyme, as shown in FIG. 3. To describe morespecifically, in the method shown in FIG. 3, first, step 1 a to step 1 dof the method shown in FIG. 1 are carried out to amplify a genomic DNAfragment having an adaptor attached to the both ends (Step 3 a to 3 d).Then, the amplified genomic DNA fragment is digested with a restrictionenzyme (hereinafter, restriction enzyme C) having a differentrecognition sequence from the restriction enzymes used in step 3 b (Step3 e). Owing to this step, the PCR fragment amplified in step 3 d isdigested into further shorter fragments.

In this manner, more than one region covering at least a part of thegenomic DNA fragments obtained by digesting genomic DNA with restrictionenzyme A and restriction enzyme B can be specified by more than one DNAfragment without sequencing. As the region to be specified, thosehaving, for example, a 20 to 100 nucleotide length, preferably a 30 to90 nucleotide length, and more preferably, a 50 to 75 nucleotide lengthare mentioned, as mentioned above. In other words, as restriction enzymeC, one capable of cleaving a genomic DNA fragment obtained by digestinggenomic DNA with restriction enzyme A and restriction enzyme B into DNAfragments having, for example, a 20 to 100 nucleotide length, preferablya 30 to 90 nucleotide length, and more preferably a 50 to 75 nucleotidelength can be used.

Next, the DNA fragments obtained by digestion with restriction enzyme Care separated from type to type to obtain probes (Step 3 f). In thisstep, the DNA fragments obtained by digesting with restriction enzyme Ccan be separated by electrophoresis, followed by cutting out.Furthermore, the separated DNA fragment may be further cloned into avector and used as a probe, or after cloning, may be further amplifiedand used as a probe. Also in this method, more than one probe respondsto the genomic DNA fragments obtained in step 3 d from genomic DNAderived from a predetermined organism.

DNA Microarray

The DNA microarray having a probe designed as mentioned above can beprepared by a method known in the art. For example, a DNA microarrayhaving a probe designed by the method shown in FIG. 1 or FIG. 2 can beprepared by synthesizing an oligonucleotide having a desired nucleotidesequence on a carrier based on the nucleotide sequence of each probedesigned by the method shown in FIG. 1 or FIG. 2. Herein, the method forsynthesizing an oligonucleotide is not particularly limited and a methodknown in the art can be applied. For example, a method of synthesizingan oligonucleotide on a carrier by a photolithographic technology incombination with a light irradiation chemosynthesis technique can beapplied. As another method that can be applied, an oligonucleotidehaving a linker molecule, which has a high affinity for a carriersurface, on an end may be separately synthesized based on the nucleotidesequence data of each probe designed by the method shown in FIG. 1 orFIG. 2, and thereafter immobilized at a predetermined position on thecarrier surface.

Furthermore, the probe designed and prepared by the method shown in FIG.3 is immobilized on a carrier to prepare a DNA microarray having a probedesigned by the method shown in FIG. 3. In this case, for example, theprobe designed by the method shown in FIG. 3 is spotted on a carrier bya pin type arrayer and a nozzle type arrayer to prepare a DNAmicroarray.

The DNA microarray prepared as described above has one or more probeshaving a nucleotide length shorter than the genomic DNA fragment, whichis obtained by a restriction enzyme treatment of genomic DNA derivedfrom a predetermined organism. More specifically, the DNA microarrayprepared as described above is used in detecting a predetermined genomicDNA fragment by one or more probes having a nucleotide length shorterthan the genomic DNA fragment. Particularly, the DNA microarraypreferably has more than one probe to a predetermined genomic DNAfragment to detect the genomic DNA fragment by the more than one probe.

Note that, as the DNA microarray, any type of microarray may be usedsuch as a microarray using a flat surface substrate formed of e.g.,glass or silicone as a carrier, a beads array using micro beads as acarrier or a three dimensional microarray having a probe immobilized onthe inner surface of a hollow fiber.

Method for Detecting Mutation

A mutation present in genomic DNA can be detected by using the DNAmicroarray prepared as described above. The mutation herein refers to apolymorphism such as a single polymorphism present between homogenousorganisms, a variation of a nucleotide sequence present between relatedspecies or a mutation artificially introduced into a predeterminedorganism.

More specifically, first, a genomic DNA is extracted from an organism tobe tested, as shown in FIG. 4. The organism to be tested herein is anorganism to be compared to the organism used in preparing the DNAmicroarray. Then, the extracted genomic DNA is digested with therestriction enzyme used in preparing the DNA microarray to prepare morethan one genomic DNA fragment. Subsequently, the obtained genomic DNAfragments are connected to the adaptor used in preparing the DNAmicroarray. Next, the genomic DNA fragment having an adaptor attached tothe both ends is amplified by use of a primer used in preparing the DNAmicroarray. In this manner, the genomic DNA derived from an organism tobe tested, which corresponds to the genomic DNA fragment amplified instep 1 d for preparing the DNA microarray, the genomic DNA fragmentwhose nucleotide sequence is specified in step 2 a, and the genomic DNAfragment amplified in step 3 d, can be amplified.

In this step, of the genomic DNA fragments having an adaptor addedthereto, a predetermined genomic DNA fragment may be selectivelyamplified. For example, in the case where more than one adaptor is usedso as to correspond to more than one restriction enzyme, the genomic DNAfragment having a specific adaptor added thereto can be selectivelyamplified. Furthermore, of the genomic DNA fragments obtained bydigesting genomic DNA with more than one restriction enzyme, only to agenomic DNA fragment having a protruding end corresponding to apredetermined restriction enzyme, an adaptor is added. In this manner, agenomic DNA fragment having the adaptor added thereto can be selectivelyamplified. Likewise, a predetermined genomic DNA fragment can beconcentrated by selectively amplifying it.

Next, a label is added to the amplified genomic DNA fragment. As thelabel, any substance may be used as long as it is known in the art. Asthe label, for example, a fluorescent molecule, a pigment molecule and aradioactive molecule can be used. Note that, this step may be omitted byperforming the step of amplifying a genomic DNA fragment by usingnucleotides having a label. This is because an amplified DNA fragment islabeled by amplifying the genomic DNA fragment by use of a nucleotidehaving a label in above step.

Next, the genomic DNA fragment having a label is brought into contactwith a DNA microarray under predetermined conditions to hybridize theprobe immobilized to the DNA microarray with the genomic DNA fragmenthaving a label. At this time, the probe partly hybridizes with thegenomic DNA fragment under highly stringent conditions under which theprobe does not hybridize if a single nucleotide mismatch is present butonly hybridizes if the nucleotides completely match with each other.Under such highly stringent conditions thus employed, a small mutationsuch as a single polymorphism can be detected.

Note that, the stringent conditions can be controlled by a reactiontemperature and a salt concentration. More specifically, further higherstringent conditions can be obtained by increasing the temperature andfurther higher stringent conditions can be obtained by reducing a saltconcentration. For example, when a probe having a 50 to 75 nucleotidelength is used, further higher stringent conditions are prepared ifconditions of 40 to 44° C., 0.21SDS, 6×SSC are employed.

Furthermore, hybridization between the probe and the genomic DNAfragment having a label can be detected based on the label. Morespecifically after a hybridization reaction between the aforementionedgenomic DNA fragment having a label and a probe, unreacted genomic DNAfragments etc. were washed away. Thereafter, the label of the genomicDNA fragment specifically hybridized to the probe is observed. Forexample, in the case where the label is a fluorescent substance, thefluorescent wavelength is detected. In the case where the label is apigment molecule, the wavelength of the pigment is detected. Morespecifically, a fluorescent detection apparatus and an image analyzeretc., usually used for DNA microarray analysis, can be used.

Particularly, if the aforementioned DNA microarray is used, the genomicDNA fragment derived from an organism to be tested is detected by one ormore probes having a shorter nucleotide length than the genomic DNAfragments. In a conventional DArT method (FIG. 9), since a genomic DNAfragment derived from a predetermined organism is amplified by PCR andused as a probe, even if a genomic DNA fragment derived from an organismto be tested having a mismatch of several tens of nucleotides, the probeoften hybridized with it (pseudo-positive reaction). However, in theaforementioned DNA microarray, since detection was made by use of one ormore probes having a shorter nucleotide length than the genomic DNAfragments, an incident probability of such a pseudo-positive reactioncan be reduced, with the result that a genomic DNA fragment derived froman organism to be tested can be highly accurately detected.Particularly, when a genomic DNA fragment derived from an organism to betested is detected by more than one probe, a small mutation contained ina genomic DNA fragment derived from an organism to be tested can bedetected by detecting the presence or absence of hybridization in morethan one probe.

Furthermore, in the aforementioned DNA microarray, an unknown mutationcan be detected. In a conventional DNA microarray using anoligonucleotide synthesized on a carrier as a probe for mutationdetection, a detection target is only a known mutation having knownsequence data. However, according to the aforementioned method fordesigning a probe, even if a genomic DNA fragment contains a mutationwhose sequence has not yet been found, such an unknown mutation can be atarget of detection. In other words, an unknown mutation can be found byuse of the DNA microarray having the aforementioned probe.

As described in the foregoing, according to the DNA microarray of thepresent invention, since a mutation contained in the genomic DNA of anorganism to be tested can be detected in comparison with that of apredetermined organism used in preparing the DNA microarray, forexample, diversity in homogeneous organisms can be analyzed at a genelevel. Furthermore, if the DNA microarray according to the presentinvention is prepared with respect to various types of variantscontained in homogenous organism, which variant an organism to be testedbelongs to can be analyzed at a gene level.

EXAMPLES

Now, the present invention will be more specifically described by way ofExamples. The technical range of the present invention is not limited bythe following Examples.

Example 1

In this Example, it was shown that a mutation present in an allele ofeach of sugar cane varieties NiF8 and Ni9 can be detected by designing aprobe in accordance with the procedure shown in FIG. 1 without using thewhole sequence data or mutation data.

(1) Material

Sugar cane varieties NiF8 and Ni9 were used.

(2) Treatment with Restriction Enzyme

Genomic DNA was extracted separately from sugar cane varieties NiF8 andNi9 in accordance with a customary method. Genomic DNA (750 ng) wastreated with restriction enzyme PstI (NEB Inc. 25 units) at 37° C. for 2hours, followed with restriction enzyme BstNI (NEB Inc., 25 units) at60° C. for 2 hours.

(3) Adaptor Ligation

To the genomic DNA fragment (120 ng) treated in the step (2), PstIsequence adaptors (5′-CACGATGGATCCAGTGCA-3′ (SEQ ID NO: 1),5′-CTGGATCCATCGTGCA-3′ (SEQ ID NO: 2)) and T4 DNA Ligase (NEB Inc., 800units) were added and a treatment was performed at 16° C., all night andall day. In this manner, the adaptor was selectively added to thegenomic DNA fragment having a PstI recognition sequence at the bothends, among those treated in the step (2).

(4) PCR Amplification

To the genomic DNA fragments (15 ng) having an adaptor obtained in thestep (3), a PstI sequence adaptor recognizing primer(5′-GATGGATCCAGTGCAG-3′ (SEQ ID NO: 3)) and Taq polymerase (companyTAKALA, PrimeSTAR, 1.25 units) were added and genomic DNA fragments wereamplified by PCR (a cycle consisting of 10 seconds at 98° C., 15 secondsat 55° C., and 1 minute at 72° C. was repeated 30 times and the PCRsample was treated at 72° C. for 3 minutes and stored at 4° C.).

(5) Acquisition of Genomic Sequence

The genomic DNA fragment amplified by PCR in the step (4) was analyzedby the Sanger method for sequencing. As a result, 2 types of genomicsequence data (A_(—)1 (SEQ ID NO: 4) and B_(—)1 (SEQ ID NO: 5)) derivedfrom NiF8 were obtained. Furthermore, genomic sequence data (A_(—)2 (SEQID NO: 6) and B_(—)2 (SEQ ID NO: 7)) of locus region of Ni9 allele wereobtained by use of sequence data of genomic sequence A_(—)1 and B_(—)1.

(6) Probe Designing

Based on the genomic sequence data (A_(—)1, B_(—)1) of the step (5), 5and 6 probes of a 50 to 70 bp were separately designed. Morespecifically, in this Example, a probe of sugar cane variety NiF8 wasdesigned. A_(—)1 and A_(—)2 alignments and the position of the designedprobe are shown in FIG. 5. Furthermore, B_(—)1 and B_(—)2 alignments andthe position of the designed probe are shown in FIG. 6.

(7) Preparation of Array

Based on the nucleotide sequence data of the designed probes, the DNAmicroarrays having these probes were prepared (outsource to Roche).

(8) Sample Preparation

Fragments from sugar cane varieties NiF8 and Ni9 were separatelyamplified by PCR in accordance with the aforementioned methods (2) to(4). PCR amplification fragments were purified by a column (company,Qiagen), and thereafter, Cy3-labeled 9mers (TriLink Inc., 1O.D.) wasadded. The mixture was treated at 98° C. for 10 minutes and allowed tostand still on ice for 10 minutes. Thereafter, Klenow (NEB Inc., 100units) was added. The mixture was treated at 37° C. for 2 hours and thenprecipitated with ethanol to prepare a labeled sample.

(9) Detection of Hybridization Signal

Hybridization was performed by use of the DNA microarray prepared in thestep (7) and using the labeled sample of the step (8) in accordance withthe NimbleGen Array User's Guide to detect a signal derived from thelabel.

(10) Calculation of Mutation Rate

A mutation rate was calculated based on homology of the genome sequenceof the loci regions of NiF8 and Ni9 alleles within respective probes.

(11) Calculation of signal intensity ratio

The signal intensity ratio is obtained by dividing the signal intensityof array using NiF8 as a sample by the signal intensity of the arrayusing Ni9 as a sample.

(12) Results and Discussion

The measurement results of signal intensity and the signal intensityratio calculated from the results are shown in Table 1 and Table 2.

TABLE 1 probe signal length Mutation rate Signal intensity intensity(bp) (%) NiF8 Ni9 ratio PA_1 60 91.7% 2,304 225 10.2 PA_2 50 54.0% 1,318249 5.3 PA_3 65 0.0% 4,837 4,554 1.1 PA_4 58 3.4% 1,738 894 1.9 PA_5 600.0% 4,240 3,075 1.4

TABLE 2 Signal Probe length Mutation rate Signal intensity intensity(bp) (%) NiF8 Ni9 ratio PB_1 69 4.3% 1,921 298 6.4 PB_2 69 10.1% 3,398272 12.5 PB_3 70 5.7% 541 247 2.2 PB_4 50 30.0% 608 209 2.9 PB_5 52 1.9%1,463 902 1.6 PB_6 70 1.4% 2,807 2,665 1.1

From FIG. 5, it was found that a single insertion/deletion mutation of101 bp and three mutations of 1 to several-bases mutation are presentbetween A_(—)1 and A_(—)2. From Table 1, it was found that, in a probe(PA_(—)3 and PA_(—)5, mutation rate 0%) having no mutation between NiF8and Ni9, high signal intensity was detected in each of NiF8 and Ni9.This means that A_(—)1 and A_(—)2 sequences corresponding to thesequence data are present respectively in the samples of NiF8 and Ni9.Furthermore, since the signal intensity ratio of both samples is as lowas 1.1 to 1.4, signal intensity ratio of a probe having no mutation waslow.

On the other hand, as a mutation rate increases, the signal intensityratio of both samples increased (1.9 (PA_(—)4) to 10.2 (PA_(—)1)). Thisis because, A_(—)2 sequence is present in the Ni9 sample but a mutationis present in A_(—)2 sequence, which corresponds to PA_(—)1, PA_(—)2,and PA_(—)4 probes, with the result that hybridization strengthdecreases and the signal of Ni9 decreases.

Similarly, from FIG. 6, it is found that three insertion/deletionmutations and 14 SNPs are present between B_(—)1 and B_(—)2. Also withrespect to B_(—)1 and B_(—)2, a signal intensity ratio increases as amutation rate increases (1.1 (PB_(—)6) to 12.5 (PB_(—)2)) as is apparentfrom Table 2.

From the above results, it was demonstrated that DNA mutation of aseveral-bp level can be detected and the site of a mutation ofseveral-tens of nucleotides can be specified by using a probe having anucleotide length shorter than a genomic DNA fragment serving as asample.

Example 2

In this Example, to the probe derived from NiF8 prepared in Example 1, amutation was artificially introduced. Based on the mutation introductionrate and the signal intensity ratio thereof to an original probe, themutation detection ability was evaluated.

(1) Material

Sugar cane variety NiF8 was used.

(2) Acquisition of Basic Probe Sequence Data

A PCR amplification fragment of NiF8 was prepared in accordance with thesteps (2) to (4) of Example 1 and the genomic sequence was determined bythe Sanger method. Based on independent genomic sequence data, 6 basicprobes having a 50 to 75 bp were prepared (Table 3).

TABLE 3 Sequence Probe Sequence length PC_1gccgtcgctcacaaggaccaacgaacggaaaggcatgcatgcagag 64 agtt (SEQ ID NO: 8)PC_2 tatgagctatatgtaatgtaagtgtactactctcctgtcaccttgc 71acttgacagca (SEQ ID NO: 9) PC_3cctctctttgctccgaaattggtcatgtactcatgttatatgcaat 78atatacggagtagtact (SEQ ID NO: 10) PC_4tcagaaacgcaacattctgcactctgattttactatatgcatcgcttctcattttactgacttg (SEQ ID NO: 11) 79 PC_5aagtaatgttatcaatcggcaaatcaaatatggccagaatcaacat 88aagaaactgagatttggcacagaaatg (SEQ ID NO: 12) PC_6ttcatctacatttagtactccatgcatatatcgcaagtttgatgtg 90acggaaatcttttgtttgcacaatacttt (SEQ ID NO: 13)

(3) Preparation of Mutation Probe

Probes were prepared by separately inserting, deleting and substitutingwith 1, 2, 3, 4, 5, 10, 15, 20 and 25 nucleotides into, from and for thebasic probes of the step (2).

(4) Array Preparation, Labeling, Hybridization-Signal Detection

A DNA microarray was prepared in the same manner as in the steps (7) to(9) of Example 1. A sample was prepared and a hybridization reaction andthe following signal detection were performed.

(5) Calculation of Signal Intensity Ratio

The value of the signal intensity ratio was obtained by dividing thesignal intensity of a mutation probe by the basic probe signalintensity. A graph and an approximation curve were prepared by Excel2007.

(6) Results and Discussion

The relationship between the mutation rate introduced into a probe andthe signal intensity detected is shown in FIG. 7. As shown in FIG. 7,the mutation rate of a probe and the signal intensity ratio are highlycorrelated (y=0.0804x−0.518, R2=0.8068). From the correlation, it wasfound that a signal intensity ratio tends to reduce to 50% or less at amutation rate of 3% or more. Even if there is a 1 bp mutation, thesignal intensity ratio decreases up to less than 50% depending upon theprobe. From the above results, it was demonstrated that mutation of asingle to several nucleotides or more can be highly accurately detectedby a probe having a nucleotide length shorter than the genomic DNAfragment.

Example 3

In this Example, using sugar cane varieties NiF8 and Ni9 genomicsequence data (5,848 nucleotides), 5 to 15 probes consisting of severaltens of bps were prepared for each genomic sequence datum and detectionof a mutation between both samples was carried out.

(1) Material

Sugar cane varieties NiF8 and Ni9 were used.

(2) Acquisition of Genomic Sequence Data

Fragments of NiF8 and Ni9 were amplified by PCR according to the steps(2) to (4) of Example 1 and analyzed by the Sanger method to obtain thegenomic sequence data. More specifically genomic sequence data of 5,848PCR amplification fragments were obtained.

(3) Preparation of Probe

Five to fifteen probes each having 50 to 75 bp were designed based onthe genomic sequence data obtained in the step (2). More specifically,based on the genomic sequence data (5,848 data), 59,462 probes weredesigned.

(4) Array Preparation, Labeling, Hybridization-Signal Detection

A DNA microarray was prepared in accordance with the steps (7) to (9) ofExample 1. A sample was prepared and a hybridization reaction and thefollowing signal detection were performed

(5) Detection of Mutation-Site Probe

When a signal intensity ratio of an array using Ni9 as a sample to anarray using NiF8 as a sample is twice or more or ½ or less, the probewas determined as a mutation-site probe.

(6) Ratio of Mutation-Site Probe Per Sequence Data

A value obtained by dividing the number of mutation-site probes persequence datum by the number of probes prepared per sequence datum, wasused.

(7) Results and Discussion

59,462 probes were designed from 5,848 genomic sequence data. Of them,the number of probes in the case where a signal intensity ratio wasbeyond 2, was 5,596. Sequence data having at least one of such a probewas 1,497. Of these sequence data, the number of data providing a signalintensity ratio of 2 or more in all probes were 189, which was 12.6% ofthe total (FIG. 8). It was considered that mutation within the sequencedata is caused by a large insertion/deletion of several kbp within arestriction enzyme recognition sequence. On the other hand, the sequencedata in which a mutation was detected in a part of probes was 87.4% ofall data. This is because an ability to detect a mutation is improved bydesigning more than one probe of several tens of bps in the interior.From the above results, in all probes, the sequence data in which amutation of this time was detected is 7.9 fold as large as the sequencedata providing a signal intensity ratio of 2 fold or more. Thus, it wasclearly demonstrated that the ability to detect a mutation improves bydesigning more than one probe having several tens of bps, which isshorter than a genomic DNA fragment serving as a sample.

Example 4

In this Example, to validate availability of a DNA microarray having aprobe designed based on known sequence data of another organism, a DNAmicroarray having a probe designed based on the total sequence data ofSorghum was prepared and a mutation of sugar cane genomic DNA wasdetected.

(1) Material

Sugar cane varieties NiF8 and Ni9 were used.

(2) Acquisition of Sorghum genomic sequence data from genome DB

From Sorghum total genomic sequence data of genome DB (Gramene:http://www.gramene.org/), sequence data between PstI recognitionsequences were obtained.

(3) Preparation of Probe

Based on the sequence data of step (2), a probe having 50 to 75 bp wasdesigned.

(4) Array preparation, labeling, hybridization-signal detection

A DNA microarray was prepared in accordance with the steps (7) to (9) ofExample 1. A sample was prepared and a hybridization reaction and thefollowing signal detection were performed.

(5) Calculation of the Number of Mutation-Site Probes

When a signal intensity ratio of an array using Ni9 as a sample to anarray using NiF8 as a sample is twice or more or ½ or less, the probewas determined as a mutation-site probe.

(6) Results and Discussion

In this Example, 1,744,104 probes were designed based on Sorghum genomicsequence data, as shown in Table 4.

TABLE 4 Number of probes Total number of test Signal Detection numberChromosome samples (1,000 or more) of mutations Chr. 1 215,534 14,9883,959 Chr. 2 191,280 12,627 3,383 Chr. 3 214,387 13,138 3,629 Chr. 4183,499 10,658 2,794 Chr. 5 161,513 6,810 1,952 Chr. 6 164,830 8,8302,330 Chr. 7 161,463 6,846 1,959 Chr. 8 138,922 5,819 1,656 Chr. 9153,484 7,426 1,930 Chr. 10 159,192 8,278 2,155 All 1,744,104 95,42025,747

Of them, the number of sequence data having a probe providing a signalintensity of 1,000 or more was 95,420. The ratio of this to the numberof sequence data used was 4.2% to 7.0% per Sorghum chromosome. In total,it was 5.5%. From the results, it was considered that a homologousregion to these probe sequences is present each in sugar cane NiF8 andNi9. Furthermore, of these probes, the number of probes in the casewhere a signal intensity ratio was beyond 2, in NiF8 and Ni9, was25,747. It was 1.2% to 1.8% per chromosome of the test probes. In total,it was 1.5%. In the region of a probe providing a signal intensity ratioexceeding 2, it is considered that a mutation is present between NIF8and Ni9. From the results in the foregoing, it is clearly demonstratedthat designing a probe by use of genome information of another organismcan be used for analyzing gene mutation in a predetermined organism.

All publications and patents and patent applications cited in thespecification are incorporated by reference in its entirety.

1-20. (canceled)
 21. A method for designing a probe, comprising thesteps of: specifying one or more regions having a shorter nucleotidelength than genomic DNA fragment flanked by restriction enzymerecognition sites, contained in genomic DNA derived from a targetorganism, and covering at least one portion of the genomic DNA fragment;and designing the specified one or more regions as a probe for detectinga mutation contained in the genomic DNA fragment.
 22. The method fordesigning a probe according to claim 21, wherein the one or more regionsis specified by performing the following steps: (1 a) extracting thegenomic DNA; (1 b) digesting the extracted genomic DNA with therestriction enzyme; (1 c) connecting an adaptor to the genomic DNAfragments obtained the step (1 b); (1 d) amplifying the genomic DNAfragments using a primer capable of hybridizing to the adaptor; (1 e)sequencing the amplified genomic DNA fragments; and (1 f) determiningthe one or more regions based on the nucleotide sequence.
 23. The methodfor designing a probe according to claim 22, wherein, in the step (1 b),the genomic DNA is digested with more than one restriction enzyme. 24.The method for designing a probe according to claim 23, wherein, in thestep (1 c), an adaptor is connected corresponding to one restrictionenzyme selected from the more than one restriction enzyme orcorresponding to a part of the more than one restriction enzyme used.25. The method for designing a probe according to claim 22, wherein theadaptor has a complementary sequence to a protruding end of the genomicDNA fragments obtained in the step (1 b).
 26. The method for designing aprobe according to claim 21, wherein the one or more regions arespecified using the nucleotide sequence data on the genomic DNA byperforming the following steps: (2 a) searching the nucleotide sequencedata on the genomic DNA for the restriction enzyme recognition sequenceto specify the nucleotide sequence of the genomic DNA fragments obtainedby digesting the genomic DNA with the restriction enzyme; and (2 b)determining the one or more regions based on the specified nucleotidesequence.
 27. The method for designing a probe according to claim 26,wherein, in the step (2 a), the genomic DNA fragments obtained bydigesting the genomic DNA with more than one restriction enzyme aresequenced.
 28. The method for designing a probe according to claim 27,wherein, in the step (2 b), the one or more regions are determined withrespect to the genomic DNA fragments flanked by one restriction enzymeselected from the more than one restriction enzyme or a part of morethan one restriction enzyme used.
 29. The method for designing a probeaccording to claim 21, wherein the one or more regions are determined byperforming the following steps: (3 a) extracting the genomic DNA; (3 b)digesting the extracted genomic DNA with the restriction enzyme; (3 c)connecting an adaptor to the genomic DNA fragments obtained in the step(3 b); (3 d) amplifying the genomic DNA fragments using a primer capableof hybridizing to the adaptor; (3 e) digesting the amplified genomic DNAfragment with another restriction enzyme; and (3 f) separating the DNAfragments obtained by digestion in the step (3 e) as probes.
 30. Themethod for designing a probe according to claim 29, wherein, in the step(3 b), the genomic DNA is digested with more than one restrictionenzyme.
 31. The method for designing a probe according to claim 30,wherein, in the step (3 c), an adaptor is connected corresponding to onerestriction enzyme selected from the more than one restriction enzyme orcorresponding to a part of the more than one restriction enzyme used.32. The method for designing a probe according to claim 29, wherein, theadaptor has a complementary sequence to a protruding end of the genomicDNA fragments obtained in the step (3 b).
 33. The method for designing aprobe according to claim 21, wherein the designed probe has a 20 to 100nucleotide length.
 34. A DNA microarray comprising a probe designed bythe method for designing a probe according to claim 21 and a carrier onwhich the probe to be immobilized.
 35. The DNA microarray according toclaim 34, wherein the probe is synthesized on the carrier based on thesequence data.
 36. A method for detecting a mutation using a DNAmicroarray, comprising the steps of: extracting a genomic DNA derivedfrom an organism to be tested; digesting the genomic DNA with arestriction enzyme having the same recognition sequence as therestriction enzyme used in the designing a probe immobilized on the DNAmicroarray according to claim 34; connecting an adaptor to the genomicDNA fragments obtained by the restriction enzyme treatment; amplifyingthe genomic DNA fragments using a primer capable of hybridizing to theadaptor; and detecting a hybrid of the genomic DNA fragment with theprobe by bringing the amplified genomic DNA fragment into contact withthe DNA microarray according to claim
 34. 37. The method for detecting amutation using the DNA microarray according to claim 36, wherein, in thestep of digesting the genomic DNA, the genomic DNA is digested with morethan one restriction enzyme.
 38. The method for detecting a mutationusing a DNA microarray according to claim 37, wherein, in the step ofconnecting an adaptor is connected corresponding to one restrictionenzyme selected from the more than one restriction enzyme orcorresponding to a part of the more than one restriction enzyme areconnected.
 39. The method for detecting a mutation using a DNAmicroarray according to claim 36, wherein the adaptor has acomplementary sequence to a protruding end of the genomic DNA fragmentsobtained in the step of digesting the genomic DNA with a restrictionenzyme.
 40. The method for detecting a mutation using a DNA microarrayaccording to claim 36, wherein the organism to be tested is differentfrom the organism used in preparing the DNA microarray.